Difference between revisions of "Part:BBa K1139150:Experience"
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| + | <partinfo>BBa_K1139150 short</partinfo> | ||
| + | __TOC__ | ||
| + | ===Materials and Methods=== | ||
| + | <b>1. Construction</b><br> | ||
| + | -pSB6A1-Ptet-GFP (N99)…positive control<br> | ||
| + | -pSB6A1-promoterless-GFP (N99)…negative control<br> | ||
| + | -pSB6A1-PRM/lac-GFP (N99)…sample with CI<br> | ||
| + | -pSB6A1--PRM/lac-GFP (JM2.300)…sample without CI<br> | ||
| + | This N99 strain expresses CI from its genome constitutively.<br> | ||
| − | + | [[Image:titech2013_parts_K1139150_exp_Fig1.jpg|frameless|none|250px||]] | |
| − | + | ||
| − | + | <b>2. Assay protocol</b><br> | |
| + | 1. Prepare overnight cultures of each cell at 37°C for 12 hours.<br> | ||
| + | 2. Take 30 µL of the overnight cultures into LB (3 mL) containing antibiotics (Amp 50 µg/mL) and 1 mM of IPTG* (=> fresh culture)<br> | ||
| + | *We added IPTG in order to make sure to repress the expression of LacI derived from <i>E. coli</i> genome.<br> | ||
| + | 3. After 4 hours of induction, measure the fluorescence intensity with a flow cytometer.<br> | ||
| + | |||
| + | ===Results=== | ||
| + | Fig. 1 shows the fluorescence intensity detected by flow cytometer.<br> | ||
| + | Fig. 2 is the extracted data which shows the comparison of N99 (IPTG+, with constitutive CI) and JM2.300 (IPTG+, without CI).<br> | ||
| + | |||
| + | [[Image:titech2013_parts_K1139150_Fig2.jpg|thumb|left|310px|<b>Fig. 1.</b> Fluorescence intensity detected by flow cytometer]] | ||
| + | [[Image:titech2013_parts_K1139150_Fig3.jpg|thumb|none|240px|<b>Fig. 2.</b> Comparison of N99 and JM2.300]] | ||
| + | |||
| + | <br> | ||
| + | |||
| + | ===Discussion=== | ||
| + | N99 cells (CI+) showed higher fluorescence intensity than that of JM2.300 cells (CI-). From this result, we assume that our <i>RM/lac</i> hybrid promoter was actually activated by CI. In addition, N99 (IPTG-) showed lower fluorescence than that of N99 (IPTG+). From this result, we can assume that our <i>RM/lac</i> hybrid promoter was repressed by LacI derived from the <i>E. coli</i> genome. | ||
| + | |||
| + | For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/RM-lac_Hybrid_Promoter_Assay our work in Tokyo_Tech 2013 wiki]. | ||
===Applications of BBa_K1139150=== | ===Applications of BBa_K1139150=== | ||
Latest revision as of 21:45, 28 October 2013
Prm/lac-GFP-TT
Contents
Materials and Methods
1. Construction
-pSB6A1-Ptet-GFP (N99)…positive control
-pSB6A1-promoterless-GFP (N99)…negative control
-pSB6A1-PRM/lac-GFP (N99)…sample with CI
-pSB6A1--PRM/lac-GFP (JM2.300)…sample without CI
This N99 strain expresses CI from its genome constitutively.
2. Assay protocol
1. Prepare overnight cultures of each cell at 37°C for 12 hours.
2. Take 30 µL of the overnight cultures into LB (3 mL) containing antibiotics (Amp 50 µg/mL) and 1 mM of IPTG* (=> fresh culture)
- We added IPTG in order to make sure to repress the expression of LacI derived from E. coli genome.
3. After 4 hours of induction, measure the fluorescence intensity with a flow cytometer.
Results
Fig. 1 shows the fluorescence intensity detected by flow cytometer.
Fig. 2 is the extracted data which shows the comparison of N99 (IPTG+, with constitutive CI) and JM2.300 (IPTG+, without CI).
Discussion
N99 cells (CI+) showed higher fluorescence intensity than that of JM2.300 cells (CI-). From this result, we assume that our RM/lac hybrid promoter was actually activated by CI. In addition, N99 (IPTG-) showed lower fluorescence than that of N99 (IPTG+). From this result, we can assume that our RM/lac hybrid promoter was repressed by LacI derived from the E. coli genome.
For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/RM-lac_Hybrid_Promoter_Assay our work in Tokyo_Tech 2013 wiki].
Applications of BBa_K1139150
User Reviews
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